General Background and State of the Art: This invention is directed to an enzyme from Gram-positive bacteria, designated sortase-transamidase, nucleic acid segments encoding the enzyme, and methods of use of the enzyme.
Human infections caused by Gram-positive bacteria present a medical challenge due to the dramatic increase in multiple antibiotic resistance strains in recent years. Gram-positive bacteria that can cause serious or fatal infections in humans include Staphylococcus, Streptococcus, Enterococcus, Pneumococcus, Bacillus, Actinomyces, Mycobacterium, and Listeria, as well as others. Infections caused by these pathogens are particularly severe and difficult to treat in immunologically compromised patients. These include patients suffering from infection with the Human Immunodeficiency Virus (HIV), the virus that causes AIDS, as well as patients given immune-suppressive agents for treatment of cancer or autoimmune diseases. In particular, infections caused by various Mycobacterium species, including M. tuberculosis, M. bovis, M. avium, and M. intracellulare, are frequently the cause of disease in patients with AIDS.
Therefore, it is apparent that new target sites for bacterial chemotherapy are needed if such pathogenic organisms are to be controlled.
A unique characteristic of these pathogens and many Gram-positive bacteria is their surface display of proteins anchored to the cell wall. In fact, many of these molecules are known to be involved in essential cellular functions, including pathogenesis in a susceptible host. Thus, a possible disruption in this anchoring process may prove to be an effective treatment against these disease-causing elements.
The anchoring of surface molecules to the cell wall in Gram-positive bacteria has been demonstrated to involve a conserved pathway, culminating in recognition of a conserved cleavage/anchoring site by some previously uncharacterized cellular machinery. Molecules whose ultimate location is the cell wall must invariably be translocated across the single cellular membrane of these organisms. This is mediated for all cell wall anchored proteins by the well studied secretory pathway, involving cleavage of an amino-terminal signal peptide by a type I signal peptidase. Upon translocation of the molecule out of the cytoplasm, a mechanism must be present that extracellularly recognizes this protein as a substrate for anchoring. This process has been previously shown to involve the carboxyl-terminally located cell wall sorting signal, consisting of a highly conserved motif such as LPXTG (SEQ ID NO:1), in which X can represent any of the twenty naturally occurring L-amino acids, followed by a series of hydrophobic residues and ultimately a sequence of positively-charged residues. Thus, once amino-terminally modified and successfully secreted, a polypeptide with this carboxyl-terminal sequence can present itself as a substrate to be processed by the anchoring machinery. At this time, cleavage of the sorting signal after the threonine residue is coupled with covalent linkage of the remainder of the polypeptide to the free amino group of the pentaglycine crossbridge in the cell wall.
It is this transpeptidation reaction that anchors mature surface proteins to the peptidoglycan layer, from which point the molecules can serve their biological functions. Therefore, there is a need to isolate and purify the enzymes that catalyze this reaction. There is also a need to identify the genes encoding such enzymes in order that the enzymes can be produced by genetic engineering techniques.
Additionally, there is also a need to develop new methods for displaying proteins or peptides on the surfaces of bacteria. For many purposes, it is desirable to display proteins or peptides on the surfaces of bacteria so that the proteins or peptides are accessible to the surrounding solution, and can, for example, be bound by a ligand that is bound specifically by the protein or peptide. In particular, the display of proteins on the surface of bacteria is desirable for the preparation of vaccines, the linkage of molecules such as antibiotic molecules or diagnostic reagents to cells, for screening reagents such as monoclonal antibodies, and for the selection of cloned proteins by displaying the cloned proteins, then observing their reaction with specific reagents such as antibodies. One way of doing this has been with phage display (G. P. Smith, xe2x80x9cFilamentous Fusion Phage: Novel Expression Vectors that Display Cloned Antigens on the Virion Surface,xe2x80x9d Science 228:1315-1316 (1985)). However, phage display is limited in its practicality, because it requires that the protein being displayed to be inserted into a coat protein of filamentous phage and retain its activity while not distorting the conformation of the coat protein, allowing functional virions to be formed. In general, this technique is therefore limited only to small peptide and proteins.
Therefore, there is a need for a more general method of peptide and protein display.
The present invention is directed to sortase-transamidase enzymes from Gram-positive bacteria, particularly the products of the surface protein sorting genes (srtA and srtB) of Staphylococcus aureus, and methods for their use, particularly in the areas of drug screening and peptide and protein display and as targets for bacteriocidal compounds or antibiotics.
One aspect of the present invention is a substantially purified sortase-transamidase enzyme from a Gram-positive bacterium, the enzyme catalyzing a reaction that covalently cross-links the carboxyl terminus of a protein having a sorting signal to the peptidoglycan of a Gram-positive bacterium, the sorting signal having a motif of LPX3X4G therein, wherein sorting occurs by cleavage between the fourth and fifth residues of the LPX3X4G motif. Typically, the Gram-positive bacterium is a species selected from the group consisting of but not limited to Staphylococcus aureus, S. sobrinus, Enterococcus faecalis, Streptococcus pyogenes, and Listeria monocytogenes. Preferably, the Gram-positive bacterium is S. aureus, and more preferably, the enzyme is the product of the srtA gene or the srtB gene of S. aureus. 
Preferably, the enzyme has a molecular weight of about 23,539 or about 29, 076 daltons and the sorting signal further includes: (2) a substantially hydrophobic domain of at least 31 amino acids carboxyl to the motif; and (3) a charged tail region with at least two positively charged residues carboxyl to the substantially hydrophobic domain, at least one of the two positively charged residues being arginine, the two positively charged residues being located at residues 31-33 from the motif, wherein X3 is any of the twenty naturally-occurring L-amino acids and X4 is selected from the group consisting of alanine, serine, and threonine.
The enzyme includes an amino acid sequence of: (1)
and (2) sequences incorporating one or more conservative amino acid substitutions in SEQ ID NO:3, wherein the conservative amino acid substitutions are any of the following: (1) any of isoleucine, leucine, and valine for any other of these amino acids; (2) aspartic acid for glutamic acid and vice versa; (3) glutamine for asparagine and vice versa; and (4) serine for threonine and vice versa.
Alternatively, the enzyme can include an amino acid sequence of: (1)
and (2) sequences incorporating one or more conservative amino acid substitutions in SEQ ID NO:38, wherein the conservative amino acid substitutions are any of the following: (1) any of isoleucine, leucine, and valine for any other of these amino acids; (2) aspartic acid for glutamic acid and vice versa, (3) glutamine for asparagine and vice versa; and (4) serine for threonine and vice versa.
Another aspect of the present invention is a nucleic acid sequence encoding this enzyme. In one alternative, the nucleic acid sequence includes therein a sequence of:
and (2) a sequence complementary to SEQ ID NO:2 (SEQ ID NO:39). In another alternative, the nucleic acid sequence can include a sequence hybridizing with SEQ ID NO:2 or a sequence complementary to SEQ ID NO:2 with no greater than about a 15% mismatch under stringent conditions. Preferably, the degree of mismatch is less than about 5%; more preferably, the degree of mismatch is less than about 2%.
In yet another aspect of the present invention a nucleic acid sequence encoding this enzyme includes therein a sequence of:
and (2) a sequence complementary to SEQ ID NO:37 (SEQ ID NO:40). In another alternative, the nucleic acid sequence can include a sequence hybridizing with SEQ ID NO:37 or a sequence complementary to SEQ ID NO:37 with no greater than about a 15% mismatch under stringent conditions. Preferably, the degree of mismatch is less than about 5%; more preferably, the degree of mismatch is less than about 2%.
Yet another aspect of the present invention is a vector comprising a nucleic acid sequence of the present invention operatively linked to at least one control sequence that controls the expression or regulation of the nucleic acid sequence.
Yet another aspect of the present invention is a host cell transfected with a vector of the present invention.
Another aspect of the present invention is a method for producing a substantially purified sortase-transamidase enzyme. The method comprises the steps of:
(1) culturing a host cell according to the present invention under conditions in which the host cell expresses the encoded sortase-transamidase enzyme; and
(2) purifying the expressed enzyme to produce substantially purified sortase-transamidase enzyme.
Another aspect of the present invention is a method for screening a compound for anti-sortase-transamidase activity. This method is important in providing a way to screen for antibiotics that disrupt the sorting reaction and are likely to be effective in treating infections caused by Gram-positive bacteria.
In one alternative, the screening method comprises the steps of:
(1) providing a substantially purified sortase-transamidase enzyme according to the present invention;
(2) performing an assay for sortase-transamidase in the presence and in the absence of the compound; and
(3) comparing the activity of the sortase-transamidase enzyme in the presence and in the absence of the compound to screen the compound for sortase-transamidase activity.
In another alternative, the screening method comprises the steps of:
(1) providing an active fraction of sortase-transamidase enzyme from a Gram-positive bacterium;
(2) performing an assay for sortase-transamidase in the presence and in the absence of the compound; and
(3) comparing the activity of the sortase-transamidase enzyme in the presence and in the absence of the compound to screen the compound for sortase-transamidase activity.
The active fraction of sortase-transamidase activity can be a particulate fraction from Staphylococcus aureus. 
The assay for sortase-transamidase enzyme can be performed by monitoring the capture of a soluble peptide that is a substrate for the enzyme by its interaction with an affinity resin. In one alternative, the soluble peptide includes a sequence of at least six histidine residues and the affinity resin contains nickel. In another alternative, the soluble peptide includes the active site of glutathione S-transferase and the affinity resin contains glutathione. In yet another alternative, the soluble peptide includes the active site of streptavidin and the affinity resin contains biotin. In still another alternative, the soluble peptide includes the active site of maltose binding protein and the affinity resin contains amylose.
Still another aspect of the present invention is an antibody specifically binding a sortase-transamidase enzyme of the present invention.
Yet another aspect of the present invention is a protein molecule comprising a substantially purified sortase-transamidase enzyme according to the present invention extended at its carboxyl-terminus with a sufficient number of histidine residues to allow specific binding of the protein molecule to a nickel-sepharose column through the histidine residues added at the carboxyl-terminus.
Still another aspect of the present invention is a method for displaying a polypeptide on the surface of a Gram-positive bacterium comprising the steps of:
(1) expressing a polypeptide having a sorting signal at its carboxy-terminal end, the sorting signal having: (a) a motif of LPX3X4G therein; (b) a substantially hydrophobic domain of at least 31 amino acids carboxyl to the motif; and (c) a charged tail region with at least two positively charged residues carboxyl to the substantially hydrophobic domain, at least one of the two positively charged residues being arginine, the two positively charged residues being located at residues 31-33 from the motif, wherein X3 is any of the twenty naturally-occurring L-amino acids and X4 is selected from the group consisting of alanine, serine, and threonine;
(2) forming a reaction mixture including: (i) the expressed polypeptide; (ii) a substantially purified sortase-transamidase according to the present invention; and (iii) a Gram-positive bacterium having a peptidoglycan to which the sortase-transamidase can link the polypeptide; and
(3) allowing the sortase-transamidase to catalyze a reaction that cleaves the polypeptide within the LPX3X4 motif of the sorting signal and covalently cross-links the amino-terminal portion of the cleaved polypeptide to the peptidoglycan to display the polypeptide on the surface of the Gram-positive bacterium.
Another display method according to the present invention comprises:
(1) cloning a nucleic acid segment encoding a chimeric protein into a Gram-positive bacterium to generate a cloned chimeric protein including therein a carboxyl-terminal sorting signal as described above;
(2) growing the bacterium into which the nucleiracid segment has been cloned to express the cloned chimeric protein to generate a chimeric protein including therein a carboxyl-terminal sorting signal; and
(3) binding the polypeptide covalently to the cell wall by the enzymatic action of a sortase-transamidase expressed by the Gram-positive bacterium involving cleavage of the chimeric protein within the LPX3X4G motif so that the polypeptide is displayed on the surface of the Gram-positive bacterium in such a way that the polypeptide is accessible to a ligand.
Another aspect of the present invention is a polypeptide displayed on the surface of a Gram-positive bacterium by covalent linkage of an amino-acid sequence of LPX3X4 derived from cleavage of an LPX3X4G motif, wherein X3 is any of the twenty naturally-occurring L-amino acids and X4 is selected from the group consisting of alanine, serine, and threonine, the polypeptide being displayed on the surface of the Gram-positive bacterium in such a way that the polypeptide is accessible to a ligand.
Another aspect of the present invention is a covalent complex comprising:
(1) the displayed polypeptide; and
(2) an antigen or hapten covalently cross-linked to the polypeptide.
Yet another aspect of the present invention is a method for vaccination of an animal comprising the step of immunizing the animal with the displayed polypeptide to generate an immune response against the displayed polypeptide, or, alternatively, with the covalent complex to generate an immune response against the antigen or the hapten.
Still another aspect of the present invention is a method for screening for expression of a cloned polypeptide comprising the steps of:
(1) expressing a cloned polypeptide as a chimeric protein having a sorting signal at its carboxy-terminal end as described above;
(2) forming a reaction mixture including: (i) the expressed chimeric protein; (ii) a substantially purified sortase-transamidase enzyme according to the present invention; and (iii) a Gram-positive bacterium having a peptidoglycan to which the sortase-transamidase can link the polypeptide through the sorting signal;
(3) binding the chimeric protein covalently to the cell wall by the enzymatic action of a sortase-transamidase expressed by the Gram-positive bacterium involving cleavage of the chimeric protein within the LPX3X4G motif so that the polypeptide is displayed on the surface of the Gram-positive bacterium in such a way that the polypeptide is accessible to a ligand; and
(4) reacting the displayed polypeptide with a labeled specific binding partner to screen the chimeric protein for reactivity with the labeled specific binding partner.
Still another aspect of the present invention is a method for the diagnosis or treatment of a bacterial infection caused by a Gram-positive bacterium comprising the steps of:
(1) conjugating an antibiotic or a detection reagent to a protein including therein a carboxyl-terminal sorting signal as described above to produce a conjugate; and
(2) introducing the conjugate to an organism infected with a Gram-positive bacterium in order to cause the conjugate to be sorted and covalently cross-linked to the cell walls of the bacterium in order to treat or diagnose the infection.
If an antibiotic is used, typically it is a penicillin, ampicillin, vancomycin, gentamicin, streptomycin, a cephalosporin, amikacin, kanamycin, neomycin, paromomycin, tobramycin, ciprofloxacin, clindamycin, rifampin, chloramphenicol, norfloxacin, or a derivative of these antibiotics.
Similarly, another aspect of the present invention is a conjugate comprising an antibiotic or a detection reagent covalently conjugated to a protein including therein a carboxyl-terminal sorting signal as described above to produce a conjugate. In still another aspect of the present invention, a composition comprises the conjugate with a pharmaceutically acceptable carrier.
Another aspect of the present invention is a substantially purified protein having at least about 50% match with best alignment with the amino acid sequences of at least one of the putative homologous proteins of Streptococcus pyogenes (SEQ.ID NO.4), Actinomyces naeslundii (SEQ.ID NO.5), Enterococcus faecalis (SEQ.ID NO.6), Streptococcus mutans (SEQ.ID.NO.7) or Bacillus subtilis (SEQ.ID NO.8) and having sortase-transamidase activity. Preferably, the match is at least about 60% in best alignment; more preferably, the match is at least about 70% in best alignment.